1. Field of the Invention
The present invention relates to a method and system for controlling fluid flow in one or more channels of a microfluidic device used for performing biological reactions. Specifically, the present invention relates to detecting the edge between fluid slugs having markers of different colors in a channel of the microfluidic device and controlling the flow of the fluid slugs based on the edge location.
2. Discussion of the Background
Microfluidic devices are widely known and used for performing biological reactions. A number of approaches have been developed to control fluid flow in a microfluidic device. Specifically, flow control in microfluidic devices including multiple channels has been described in US Patent Application Publications No. 2014/0272927, No. 2013/0177913, No. 2012/0058460, No. 2012/0052560 the disclosures of which are hereby incorporated by reference. Many microfluidic devices known in the art include microchannels having sequentially alternating test solutions and carrier fluid. The purpose of the carrier fluid, (“blanking slugs”) is to deter transfer of material from one test solution to another. Another purpose of the carrier fluid is to provide a distinguishable transition between slugs that may be used to track and control the fluid flow in the microchannel. The carrier fluid and the test solution may include fluorescent markers to allow for detecting the edge between adjacent slugs to track and control the fluid flow in the microchannel. A detector is usually provided to detect the progress of a biological reaction and track the fluid flow in the microchannel. Specifically, the detector can independently detect signals from one or more detectable markers. Biological reactions performed in the microfluidic devices may include a polymerase chain reaction (PCR) or a nucleic acid melting analysis. Other details of fluorescence imaging systems that may be used in connection with the microfluidic systems are described in U.S. Patent Application Publication No. 2008/0003594 the disclosure of which is hereby incorporated by reference.
Methods for controlling fluid flow in a microchannel of a microfluidic device based on the position of the edge between adjacent slugs have been developed and used. For instance, Coursey et al. (US Patent Application Publication No. 2012/0058460), the disclosure of which is hereby incorporated by reference, relate to a method of color thresholding using two color planes (e.g., blue and red) to obtain two measures of slug position: one based on the position of sample slugs (e.g., blue slugs) and one based on the position of blanking slugs (e.g., the red slugs). To identify an edge between two slugs, pixels that satisfy threshold conditions in a first wavelength plane of the image and a second wavelength plane of the image are identified. The threshold is usually pre-selected by the user.
Alternatively, Coursey et al. disclose using correlation methods, such as, for example, calculating a cross-correlation and covariance with a pre-determined template image, rather than threshold detection. Identifying an edge between the first slug and the second slug comprises cross-correlating the region of interest with a predetermined template image. A simple binary template image (black on one side and white on the other) is used to detect an edge of a slug. The transition from the black side to the white side of the pre-determined template image represents the slug edge. The black/white pre-defined template image is then cross-correlated with the slug images to determine the location of the slug edge (i.e., the position at which the correlation between the template and the image data is at a maximum or minimum).
Another approach to identifying location of the edge between adjacent fluid slugs in a microchannel is based on intensity gradient across the edge and employs Otsu's method (“A threshold selection method from gray-level histograms,” IEEE Trans. Sys., Man., Cyber. 9 (1): 62-66 (1979)). Otsu's method is used to automatically perform clustering-based image thresholding. The method assumes that the image contains two classes of pixels (pixels of the first slug and pixels of the second slug) characterized by their intensities. The goal is to find an optimum intensity threshold defining the edge between the two slugs. Accordingly, Otsu's method employs image thresholding performed in intensity domain. In other words, the pixels of the image can be divided into two classes based on their intensities provided by an image histogram. The horizontal axis of the image histogram represents the intensity variations, while the vertical axis represents the number of pixels having that particular intensity. The optimum intensity threshold separating the two classes is calculated such that the intra-class variance is minimal (between-class variance is maximum). The calculated optimum intensity threshold combined with channel geometry defines the position of the edge between adjacent slugs in a channel.
Accordingly, to determine location of the edge between adjacent fluid slugs in a microchannel based on intensity gradient across the edge, the image histogram of a channel image is thresholded in the intensity domain to detect blob regions in the image. The blob regions presumably correspond to different slugs of interest. The microchannel is presumed to have a known location and orientation. The edge of the detected blobs is assumed to be the edge of interest. The detected blobs are validated against the presumed location of the channels. This approach generally works well in low noise scenarios, but its robustness can be compromised if there are bubbles or other imaging artifacts that cause broken or poorly defined blobs. Additionally, the technique may require some image pre-filtering to obtain good results, and the technique is relatively computationally expensive.
Accordingly, there is a need for a robust and simple solution that allows for detecting location of the edge between adjacent fluid slugs in a microchannel.